About Transformation

Transcription

1 About Transformation In 1928, Frederick Griffith was working on this problem of finding a vaccine against pneumonia caused by the bacteria Streptococcus pneumoniae. Here s what he found: In Experiment A, Griffith injected mice with a deadly strain of pneumococcus that had a capsule. All the mice died. When Griffith removed the capsule in Experiment B the bacteria became harmless, and all the injected mice lived. Griffith then heat-inactivated the deadly capsulated pneumococcus for Experiment C, and all the injected mice survived. Finally, in Experiment D, Griffith mixed live harmless pneumococcus with heat-killed capsulated pneumococcus. While neither of these strains killed mice alone, they did in combination! All the mice died.

2 Transform Lab Details Transformation in Nature: Recipient bacteria and loose plasmid fragments secrete chemical competence factors that allow the bacteria to take in the plasmid. Transformation in the Lab: 2 CaCl and heat/cold shock procedures are used to imitate competence factor and rener the E. coli competent or capable of taking in the plasmid DNA. These steps must be done precisely to achieve good results.

3 Our plasmid: PGLO is a genetically engineered plasmid that contains three gene groups of interest: 1. Bacterial genes for resistance to the antibiotic ampicillin (Amp). 2. Jellyfish gene for a green fluorescent protein (GFP). 3. The inducible arabinose operon, a group of bacterial genes that makes enzymes to digest the sugar arabinose. The promoter and operator remain (Ara C), but the other genes have been replaced with GFP. An arabinose food source serves as the inducer for the arabinose operon, and it is needed in order for transformed bacteria to express the GFP gene. Ampicillin is used to separate transformed from nontransformed bacteria.

4 Day1: Do the transformation MAKE SURE you understand the steps and procedure - you will have just enough time if you are efficient. Minimum time needed is 35 minutes. Each team of 3 students will prepare a control culture and a transformation culture. Follow the procedure precisely. At the end of the day, put control and transformed bacteria tubes onto 4 separate agar plates as follows: Ampicillin kills all non-transformed bacteria. Arabinose induces expression of the GFP gene. The transformed bacteria are plated onto ampicillin agar and ampicillin + arabinose agar. There should be growth on both plates. Control bacteria are plated onto plain agar and ampicillin agar. There should be growth on the plain agar but none on the ampicillin agar.

5 Day 2: Analyze your results When you put bacteria onto an agar plate, each bacteria will divide repeatedly, producing a colony of about 1 million identical cloned bacteria. You can see the colony which appears as a dot on the plate. By counting the colonies, you can determine how many bacteria you put onto the plate. You can also do a little algebra to determine the number of bacteria per ml. You then calculate the number of transformed bacteria per ìg of plasmid to find the transformation efficiency. This procedure is explained on your lab handout. We will compare the transformation efficiency of two different strains of bacteria.

6 TRANSFORMATION PROCEDURE FOR P-GLO

7 About micropipettes You MUST USE A TIP when drawing in liquid, or you will ruin the VERY EXPENSIVE micropipette. Use a fresh tip for each sample. Used tips are discarded in the special waste bins (for biological waste),not place in the regular garbage can! Micropipettes work on the principle of air pressure. The plunger can be calibrated to various volumes. You will be using a single-volume pipet that is calibrated to 10 ìl. There are two stopping points when depressing the plunger. The first stop is the calibration point. It lets you draw in 10 ìl. To draw in a sample, push to the first stop, insert the tip into the liquid, then SLOWLY release the plunger. 10 ìl will be inside the pipette tip. The second stop is used to expel the sample. To do this, place the pipette tip against the side of the tube, press down all the way, and the entire sample will be expelled. KEEP YOUR FINGER on the plunger until you withdraw your pipette tip from the liquid drop.